The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplifying (i.e., making multiple copies) target polynucleotide sequences which employ a single RNA/DNA composite primer, with the amplification optionally involving transcription.
The developments of methods for nucleic acid amplification and detection of amplification products have advanced the detection, identification, quantification and sequence analyses of nucleic acid sequences in recent years.
Nucleic acid analysis is useful for detection and identification of pathogens, detection of gene alteration leading to defined phenotypes, diagnosis of genetic diseases or the susceptibility to a disease, assessment of gene expression in development, disease and in response to defined stimuli, as well as the various genome projects. Other applications of nucleic acid amplification method are the detection of rare cells, detection of pathogens, and the detection of altered gene expression in malignancy, and the like. Nucleic acid amplification is potentially useful for both qualitative analysis, such as the detection of the presence of defined nucleic acid sequences, and quantification of defined gene sequences. The latter is useful for assessment of and amount of pathogenic sequences as well as the determination of gene multiplication or deletion, as often found in cell transformation from normal to malignant cell type.
The detection of sequence alterations in a nucleic acid sequence is important for the detection of mutant genotypes, as relevant for genetic analysis, the detection of mutations leading to drug resistance, pharmacogenomics, etc. Various methods for the detection of specific mutations include allele specific primer extension, allele specific probe ligation, and differential probe hybridization. See, for example, U.S. Pat. Nos. 5,888,819; 6,004,744; 5,882,867; 5,710,028; 6,027,889; 6,004,745; and WO US88/02746. Methods for the detection of the presence of sequence alterations in a define nucleic acid sequence, without the specific knowledge of the alteration, were also described. Some of these methods are based on the detection of mismatches formed by hybridization of a test amplification product to a reference amplification product. The presence of mismatches in such hetero-duplexes can be detected by the use of mismatch specific binding proteins, or by chemical or enzymatic cleavage of the mismatch. A method for detection of sequence alteration which is based on the inhibition of branch migration in cruciform four stranded DNA structures was recently described. See, for example, Lishanski, A. et al. Nucleic Acids Res 28(9):E42 (2000). Other methods are based on the detection of specific conformations of single stranded amplification products. The secondary structure of a single stranded DNA or RNA is dependent on the specific sequence. Sequence alterations in a test nucleic acid target relative to a reference sequence leads to altered conformation. Altered conformation of a single stranded amplification product can be detected by a change in the electrophoretic mobility of the test amplification product as compared to that of a reference amplification product. Single stranded conformation polymorphism, SSCP, is widely used for the detection of sequence alterations. See, for example, Orita M., et al. Proc Natl Acad Sci USA 86(8):2766-70 (1989); Suzuki, Y. et al. Oncogene 5(7):1037-43 (1990); and U.S. Pat. No. 5,871,697. This method is also used in microbial identification that is based on the defined changes in a specific nucleic acid sequence in different strains or species. Mutation detection using the SSCP methods mostly employs DNA amplification products, however, RNA-SSCP methods have also been described. Sequence dependent conformation of a single stranded RNA is well-documented and was shown to lead to a defined electrophoretic mobility pattern. See, for example, Sarkar et al. Nucleic Acid Research 20(4):871-878 (1992) and Gasparini et al. Hum. Genet. 97:492-495 (1996).
Although detection of the presence of a defined nucleic acid sequence, and its sequence analysis, can be carried out by probe hybridization, the method generally lacks sensitivity when low amounts of the nucleic acid sequence is present in the test sample, such as a few molecules. One solution to this obstacle was the development of methods for generation of multiple copies of the defined nucleic acid sequence, which are suitable for further analysis. The methods for generation of multiple copies of a specific nucleic acid sequence are generally defined as target amplification methods. Other methods for increasing the sensitivity of detection of hybridization analysis are based on the generation of multiple products from the hybridized probe, or probes, for example cleavage of the hybridized probe to form multiple products or the ligation of adjacent probes to form a unique hybridization dependent product. Similarly, increased sensitivity of hybridization reaction was achieved by methods for amplification of signals generated by the hybridization event, such as the method based on hybridization of branched DNA probes.
There are many variations of nucleic acid amplification, for example, exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification. An example of exponential nucleic acid amplification method is polymerase chain reaction (PCR) which has been disclosed in numerous publications. See, for example, Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Mullis K. EP 201,184; Mullis et al. U.S. Pat. No. 4,582,788; Erlich et al. EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R. et al. U.S. Pat. No. 4,683,194. Linked linear amplification is disclosed by Wallace et al. in U.S. Pat. No. 6,027,923. Examples of ligation-based amplification are the ligation amplification reaction (LAR), disclosed by Wu et al. in Genomics 4:560 (1989) and the ligase chain reaction, disclosed in EP Application No. 0320308B1. Various methods of transcription-based amplification are disclosed in U.S. Pat. Nos. 5,766,849 and 5,654,142; and also by Kwoh et al. Proc. Natl. Acad. Sci. U.S.A. 86:1173 (1989) and Ginergeras et al. WO 88/10315.
The most commonly used target amplification method is the polymerase chain reaction, PCR, which is based on multiple cycles of denaturation, hybridization of two oligonucleotide primers, each to opposite strand of the target strands, and primer extension by a nucleotide polymerase to produce multiple double stranded copies of the target sequence. Many variations of PCR have been described, and the method is being used for amplification of DNA or RNA nucleic acid sequences, sequencing, mutation analysis and others. Thermocycling-based methods that employ a single primer, have also been described. See, for example, U.S. Pat. Nos. 5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. The primer can be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No. 5,744,308. Other methods that are dependent on thermal cycling are the ligase chain reaction (LCR) and the related repair chain reaction (RCR).
Target nucleic acid amplification may be carried out through multiple cycles of incubations at various temperatures, i.e. thermal cycling, or at one temperature (an isothermal process). The discovery of thermostable nucleic acid modifying enzymes has contributed to the fast advances in nucleic acid amplification technology. See Saiki, et al. Science 239:487 (1988). Thermostable nucleic acid modifying enzymes, such as DNA and RNA polymerases, ligases, nucleases and the like, are used in both methods dependent on thermal cycling and isothermal amplification methods. Isothermal methods such as strand displacement amplification (SDA) is disclosed by Fraiser et al. in U.S. Pat. No. 5,648,211; Cleuziat et al. in U.S. Pat. No. 5,824,517; and Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992). Other isothermal target amplification methods are the transcription-based amplification methods, in which an RNA polymerase promoter sequence is incorporated into primer extension products at an early stage of the amplification (WO 89/01050), and further target sequence, or target complementary sequence, is amplified by transcription steps and digestion of an RNA strand in a DNA/RNA hybrid intermediate product. See, for example, U.S. Pat. Nos. 5,169,766 and 4,786,600. These methods include transcription mediated amplification (TMA), self-sustained sequence replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), and variations there of. See, for example, Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878 (1990); U.S. Pat. Nos. 5,766,849 (TMA); and 5,654,142 (NASBA). Other amplifications methods use template switching oligonucleotides (TSOs) and blocking oligonucleotides. For example, the template switch amplification in which chimeric DNA primer are utilized is disclosed in U.S. Pat. No. 5,679,512 and by Patel et al. Proc. Natl. Acad. Sci. U.S.A. 93:2969-2974 (1996) and blocking oligonucleotides are disclosed by Laney et al. in U.S. Pat. No. 5,679,512.
The isothermal target amplification methods do not require a thermocycler, and are thus easier to adapt to common instrumentation platform. However the previously described isothermal target amplification methods have several drawbacks. Amplification according to the SDA methods requires the presence of sites for defined restriction enzymes, which limits its applicability. The transcription base amplification methods, such as the NASBA and TMA, on the other hand, are limited by the need for incorporation of the polymerase promoter sequence into the amplification product by a primer, a process prone to result in non-specific amplification. Moreover, the mechanism of amplification of a DNA target by these transcription based amplification methods is not well-established.
Another drawback of the current amplification methods is the potential contamination test samples by amplification products of prior amplification reactions, resulting in non-target specific amplification in samples. This drawback is a well-known problem, which is the result of the power of target amplification technology, and the formation of amplification products, which are substrates for amplification. Various means for decontamination of test samples either at the end of the amplification reaction, or prior to the initiation of target amplification have been described. In additions, method for containment of the test solution by physical means, were also described. All of these solutions are cumbersome and add to the complexity of nucleic acid testing in the common laboratory setting.
Furthermore, amplification methods that use thermocycling process have an added disadvantage of long lag times which are required for the thermocycling block to reach the xe2x80x9ctargetxe2x80x9d temperature for each cycle. Consequently, amplification reactions performed using thermocycling processes require a significant amount of time to reach completion.
Therefore, there is a need for improved nucleic acid amplification methods that overcome these drawbacks. The invention provided herein fulfills this need and provides additional benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
The invention provides methods and compositions for polynucleotide amplification, as well as applications of the amplification methods.
Accordingly, in one aspect, the invention provides methods for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising: (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5xe2x80x2 with respect hybridization of the composite primer to the template; (c) extending the composite primer with DNA polymerase; (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced.
In another aspect, the invention provides methods for amplifying a target polynucleotide sequence comprising: (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (b) hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5xe2x80x2 with respect hybridization of the composite primer to the template; (c) extending the composite primer with DNA polymerase; (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement to produce displaced primer extension product; (e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, whereby multiple copies of the target sequence are produced.
Various embodiments of the composite primer used in the methods of the invention are described herein. For example, in some embodiments, the RNA portion of the composite primer is 5xe2x80x2 with respect to the 3xe2x80x2 DNA portion. In still other embodiments, the 5xe2x80x2 RNA portion is adjacent to the 3xe2x80x2 DNA portion. For the methods described herein, one or more composite primers can be used.
Various exemplary embodiments of polynucleotides comprising a termination sequence are also described herein. In some embodiments, the polynucleotide comprising a termination sequence is a template switch oligonucleotide (TSO), which may (but not necessarily) contain one or more modifications to enhance binding to template. Accordingly, in some embodiments, the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification. Examples of suitable modifications are provided herein. In some embodiments, the polynucleotide comprising a termination sequence is a blocking sequence, which, like the TSO, may contain one or more modifications to enhance binding to template. Accordingly, in some embodiments, the blocker sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocker binds more tightly to the region as compared to a blocker without the modification. Examples of suitable modifications are provided herein.
The enzymes which may be used in the methods and compositions are described herein. For example, the enzyme that cleaves RNA may be an RNaseH.
In some aspects, a TSO provides propromoter function and also comprises a region (which may or may not be adjacent to the promoter) which hybridizes to the displaced primer extension product. In other embodiments, the polynucleotide comprising the propromoter comprises a region, preferably at the 3xe2x80x2 end, which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced extension product produces a double stranded promoter from which transcription occurs. In some embodiments, the polynucleotide comprising the propromoter is a PTO.
In some embodiments of methods of the invention, a single polynucleotide effects termination of composite primer extension and provides propromoter function, said polynucleotide comprising: (a) a termination sequence that does not effect template switch under conditions wherein the termination sequence is hybridizable (e.g., hybridizes) to a target polynucleotide; (b) a propromoter sequence, wherein the propromoter sequence is not hybridizable (e.g., does not hybridize) to a target polynucleotide under conditions wherein the termination sequence is hybridizable (e.g., hybridizes) to the target polynucleotide; and (c) a sequence which is hybridizable (e.g., hybridizes) to a complementary sequence of said target polynucleotide.
The methods are applicable to amplifying any DNA target, including, for example, genomic DNA and cDNA. One or more steps may be combined and/or performed sequentially (often in any order, as long as the requisite product(s) are able to be formed).
The invention also provides methods which employ (usually, analyze) the products of the amplification methods of the invention, such as sequencing and detection of sequence alteration(s). As is understood in the art, and described herein, detection includes determining presence or absence of a sequence of interest.
Accordingly, in one aspect, the invention provides methods of sequencing a target nucleotide sequence comprising: (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5xe2x80x2 with respect hybridization of the composite primer to the template; (c) extending the composite primer with DNA polymerase and a mixture of dNTPs and dNTP analogs (which may be labeled or unlabelled), such that primer extension is terminated upon incorporation of a dNTP analog which may be labeled or unlabelled; (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced of varying lengths; (e) analyzing the product of steps (a) through (d) to determine sequence.
In another aspect, the invention provides methods for sequencing a target nucleotide sequence comprising (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3xe2x80x2 DNA portion; (b) hybridizing the template with a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5xe2x80x2 with respect to hybridization of the composite primer to the template; (c) extending the composite primer with DNA polymerase; (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement to produce displaced primer extension product; (e) hybridizing a polynucleotide comprising a propromoter at the 5xe2x80x2 end and a region which hybridizes to the displaced primer extension product under conditions such that transcription occurs from the extension product by RNA polymerase, using a mixture of rNTPs and rNTP analogs (which may be labeled or unlabelled), such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, and such that transcription is terminated upon incorporation of an rNTP analog which may be labeled or unlabelled, whereby multiple copies of the target sequence are produced of varying lengths; (f) analyzing the product of steps (a) through (e) to determine sequence.
In some aspects, the invention provides methods of characterizing, or analyzing, sequence of a target. Some aspects are based on the RNA portion of the composite primer and accordingly the results reflect information regarding the corresponding region of the target which, if complementary or of sufficient complementarity, hybridizes to the RNA portion of the composite primer. The amount of product as compared to amount of product from performing the same amplification reaction on a reference target sequence indicates the presence or absence of a sequence, which may in turn indicate presence or absence of wildtype, mutant, or allelic variants. Various sequence detection embodiments are described herein. Thus, for example, the invention provides methods of detecting a mutation in a region of a target polynucleotide sequence, comprising conducting an amplification method described herein, wherein the region of the target polynucleotide sequence corresponds to the RNA portion of the composite primer, and wherein a mutation in the target polynucleotide results in detectably fewer amplification products as compared to the amount of amplification products produced from a reference template comprising region corresponding to the RNA portion of the composite primer which does not comprise a mutation. In these embodiments, amplification by strand displacement is decreased as compared to production from a reference template which comprises a region corresponding to the RNA portion of the composite primer which does not contain the mutation (as compared to the RNA portion of the composite primer).
Thus, the invention provides methods of characterizing a sequence of interest in a target polynucleotide, said methods comprising conducting the amplification methods of the invention wherein the sequence of an RNA portion of the composite primer is known, and wherein (a) production of detectably fewer amplification products from the template as compared to the amount of amplification products from a reference template which comprises a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer; or (b) production of detectably more amplification products from the template as compared to the amount of amplification products from a reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer. In one embodiment, the sequence of an RNA portion of the composite primer comprises a wild type sequence, and the sequence of interest is characterized in determining the presence or absence of the wild type sequence. In another embodiment, the sequence of an RNA portion of the composite primer comprises a mutant sequence, and the sequence of interest is characterized in determining the presence or absence of the mutant sequence. In yet another embodiment, the sequence of an RNA portion of the composite primer comprises an allelic sequence, and the sequence of interest in characterized in determining the presence or absence of the allelic sequence.
In other aspects, the invention provides methods of detecting a mutation (or, in some aspects, characterizing a sequence) in a target polynucleotide, comprising (a) conducting an amplification method described herein; and (b) analyzing the amplified products of the method for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide. In other embodiments, the invention provides methods of detecting a mutation (or, in some aspects, characterizing a sequence) in a target polynucleotide comprising analyzing amplified products of any of the methods described herein for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide (or, in some aspects, characterizes the target sequence).
In other aspects, the invention provides methods of producing a microarray, comprising (a) conducting an amplification method described herein; and (b) attaching the amplified products onto a solid substrate to make a microarray of the amplified products. In other embodiments, microarrays are produced by attaching amplified products by any of the methods described herein onto a solid substrate to make a microarray of amplified products.
Any of these applications can use any of the amplification methods (including various components and various embodiments of any of the components) as described herein. For example, the composite primer used may have a 5xe2x80x2 RNA portion, which may be adjacent to the 3xe2x80x2 DNA portion.
The invention also provides compositions, kits, complexes, reaction mixtures and systems comprising various components (and various combinations of the components) used in the amplification methods described herein. In one aspect, for example, the invention provides compositions comprising a composite primer, said composite primer comprising a 3xe2x80x2 DNA portion and a 5xe2x80x2 RNA portion. In some embodiments, the 5xe2x80x2 RNA portion is adjacent to the 3xe2x80x2 DNA portion. In still other embodiments, the 5xe2x80x2 RNA portion is about 5 to about 20 nucleotides and the 3xe2x80x2 DNA portion is about 5 to about 15 nucleotides. In another aspect, the invention provides compositions comprising a TSO, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification. In some embodiments, the compositions of the invention comprise any composite primer described herein and a TSO. In still other embodiments, the invention provides compositions comprising any of the composite primers described herein and any blocking sequences described herein, including those containing modifications which enhance binding to template. In other embodiments, the invention provides compositions comprising any of the composite primers described herein and a PTO.
In another aspect, the invention provides compositions comprising any of the complexes (which are generally considered as intermediates with respect to the final amplification products) described herein (see also the figures for schematic depictions of these various complexes). For example, the invention provides compositions comprising a complex of (a) a template strand; and (b) a composite primer, said composite primer comprising a 3xe2x80x2 DNA portion and an RNA portion. The RNA portion may be 5xe2x80x2 as well as adjacent to the DNA portion. In some embodiments, the complex further comprises a polynucleotide comprising a termination sequence (which may be, for example, a TSO or a blocking sequence). In some embodiments, the complex further comprises a PTO.
In another aspect, the invention provides reaction mixtures (or compositions comprising reaction mixtures) which contain various combinations of components described herein. For example, the invention provides reaction mixtures comprising (a) a polynucleotide template; (b) a composite primer comprising a 3xe2x80x2 DNA portion and an RNA portion; and (c) DNA polymerase. As described herein, any of the composite primers may be in the reaction mixture (or a plurality of composite primers), including a composite primer comprises a 5xe2x80x2 RNA portion which is adjacent to the 3xe2x80x2 DNA portion. The reaction mixture could also further comprise an enzyme which cleaves RNA from an RNA/DNA hybrid, such as RNase H. A reaction mixture of the invention can also comprise any of the polynucleotides comprising termination sequences described herein, as well as a polynucleotide comprising a propromoter and a region which hybridizes to displaced primer extension product, and an RNA polymerase. A reaction mixture of the invention can also comprise a PTO.
In another aspect, the invention provides kits for conducting the methods described herein. These kits, in suitable packaging and generally (but not necessarily) containing suitable instructions, contain one or more components used in the amplification methods. For example, the invention provides kits that comprise a composite primer comprising a 3xe2x80x2 DNA portion and an RNA portion (which may be 5xe2x80x2 and may further be adjacent to the 3xe2x80x2 DNA portion). The composite primer in the kits can be any described herein. The kits can contain further components, such as any of (a) a polynucleotide comprising a termination polynucleotide sequence; (b) a polynucleotide comprising a propromoter; (c) any of the enzymes described herein, such as an enzyme which cleaves RNA from an RNA/DNA hybrid (for example, RNaseH); and (d) a polynucleotide comprising a propromoter and a region which hybridizes to displaced primer extension product.
In another aspect, the invention provides systems for effecting the amplification methods described herein. For example, the invention provides systems for amplifying a target polynucleotide sequence or its complement, comprising (a) a composite primer comprising a 3xe2x80x2 DNA portion and an RNA portion; (b) DNA polymerase; and (c) an enzyme which cleaves RNA from an RNA/DNA hybrid (such as RNaseH). The composite primer may be any (one or more) described herein, including a composite primer which comprises a 5xe2x80x2 RNA portion which is adjacent to the 3xe2x80x2 DNA portion.